The entry of calcium into cells through voltage-gated calcium channels mediates a wide variety of cellular and physiological responses, including excitation-contraction coupling, hormone secretion and gene expression (Miller, R. J., Science (1987) 235:46-52; Augustine, G. J., et al., Annu Rev Neurosci (1987) 10:633-693). In neurons, calcium channels directly affect membrane potential and contribute to electrical properties such as excitability, repetitive firing patterns and pacemaker activity. Calcium entry further affects neuronal functions by directly regulating calcium-dependent ion channels and modulating the activity of calcium-dependent enzymes such as protein kinase C and calmodulin-dependent protein kinase II. An increase in calcium concentration at the presynaptic nerve terminal triggers the release of neurotransmitter and calcium channels, which also affects neurite outgrowth and growth cone migration in developing neurons.
Calcium channels mediate a variety of normal physiological functions, and are also implicated in a number of human disorders. Examples of calcium-mediated human disorders include but are not limited to congenital migraine, cerebellar ataxia, angina, epilepsy, hypertension, ischemia, and some arrhythmias. The clinical treatment of some of these disorders has been aided by the development of therapeutic calcium channel antagonists (e.g., dihydropyridines, phenylalkyl amines, and benzothiazepines all target L-type calcium channels) (Janis, R. J. and Triggle, D. J., in Calcium Channels: Their Properties, Functions, Regulation and Clinical Relevance (1991) CRC Press, London).
Native calcium channels have been classified by their electrophysiological and pharmacological properties into T-, L-, N—, P/Q- and R-types (reviewed in Catterall, W., Annu Rev Cell Dev Biol (2000) 16:521-55; Huguenard, J. R., Annu Rev Physiol (1996) 58:329-348). T-type (or low voltage-activated) channels describe a broad class of molecules that transiently activate at negative potentials and are highly sensitive to changes in resting potential.
The L-, N— and P/Q-type channels activate at more positive potentials (high voltage-activated), and display diverse kinetics and voltage-dependent properties (Catterall, W., supra; Huguenard, J. R., supra). L-type channels can be distinguished by their sensitivity to several classes of therapeutically useful small organic molecules, including dihydropyridines (DHPs), phenylalkylamines and benzothiazepines. In contrast, N-type and P/Q-type channels are high affinity targets for certain peptide toxins produced by venous spiders and marine snails. N-type channels are blocked by the ω-conopeptides ω-conotoxin GVIA (ω-CTx-GVIA) isolated from Conus geographus and ω-conotoxin MVIIA (ω-CTx-MVIIA) isolated from Conus magus. On the other hand, P/Q-type channels are resistant to ω-CTx-MVIIA, but are sensitive to the funnel web spider peptide, ω-agatoxin IVA (ω-Aga-IVA). R-type calcium channels. are sensitive to blocking by the tarantula toxin, SNX-482.
Neuronal high voltage-activated calcium channels are composed of a large (>200 kDa) pore-forming α1 subunit that is the target of identified pharmacological agents, a cytoplasmically localized ˜50-70 kDa β subunit that tightly binds the α1 subunit and modulates channel biophysical properties, and an ˜170 kDa α2δ subunit (reviewed by Stea, A., et al., Proc. Natl. Acad. Sci. USA (1994) 91:10576-10580; Catterall, W., supra). At the molecular level, nine different α1 subunit genes expressed in the nervous system have been identified and shown to encode all of the major classes of native calcium currents (Table 1).
TABLE 1Classification of Neuronal Calcium ChannelsGeneω-AGAω-CTxω-CTxNative ClasscDNANameIVAGVIAMVIIAdihydropyridinesP/Q-typeα1ACav2.1✓———N-typeα1BCav2.2—✓✓—L-typeα1CCav1.2———✓L-typeα1DCav1.3———✓R-typeα1ECav2.3————L-typeα1FCav1.4———✓T-typeα1GCav3.1————T-typeα1HCav3.2————T-typeα1ICav3.3————
Calcium channels have been shown to mediate the development and maintenance of the neuronal sensitization processes associated with neuropathic pain, and provide attractive targets for the development of analgesic drugs (reviewed in Vanegas, H. and Schaible, H-G., Pain (2000) 85:9-18). All of the high-threshold Ca channel types, are expressed in the spinal cord, and the contributions of L-, N and P/Q-types in acute nociception are currently being investigated. In contrast, examination of the functional roles of these channels in more chronic pain conditions strongly indicates a pathophysiological role for the N-type channel (reviewed in Vanegas, H. and Schaible, H-G., supra).
Mutations in calcium channel α1 subunit genes in animals can provide important clues to potential therapeutic targets for pain intervention. Genetically altered mice null for the α1B N-type calcium channel gene have been reported by several independent groups (Ino, M., et al., Proc. Natl. Acad. Sci. USA (2001) 98:5323-5328; Kim, C., et al., Mol Cell Neurosci (2001) 18:235-245; Kim, D., et al., Neuron (2001) 31:35-45; Saegusa, H., et al., Proc. Natl. Acad. Sci. USA (2000) 97:6132-6137; Hatakeyama, S., et al., NeuroReport (2001) 12:2423-2427). The α1B N-type null mice were viable, fertile and showed normal motor coordination. In one study, peripheral body temperature, blood pressure and heart rate in the N-type gene knock-out mice were all normal (Saegusa, H, et al., supra). In another study, the baroreflex mediated by the sympathetic nervous system was reduced after bilateral carotid occlusion (Ino, M., et al., supra). In another study, mice were examined for other behavioral changes and were found to be normal except for exhibiting significantly lower anxiety-related behaviors (Saegusa, H, et al., supra). These results suggest that the N-type channel may be a potential target for mood disorders as well as pain. In all studies, mice lacking functional N-type channels exhibit marked decreases in the chronic and inflammatory pain responses. In contrast, mice lacking N-type channels generally showed normal acute nociceptive responses.
Two examples of either FDA-approved or investigational drug that act on N-type channel are gabapentin and ziconotide. Gabapentin, 1-(aminomethyl)cyclohexaneacetic acid (Neurontin®), is an anticonvulsant originally found to be active in a number of animal seizure models (Taylor, C. P., et al., Epilepsy Res (1998) 29:233-249). Subsequent work has demonstrated that gabapentin is also successful at preventing hyperalgesia in a number of different animal pain models, including chronic constriction injury (CCI), heat hyperalgesia, inflammation, diabetic neuropathy, static and dynamic mechanoallodynia associated with postoperative pain (Taylor, C. P., et al., supra; Cesena, R. M. and Calcutt, N. A. Neurosci Lett (1999) 262:101-104; Field, M. J., et al., Pain (1999) 80:391-398; Cheng, J-K., et al., Anesthesiology (2000) 92:1126-1131; Nicholson, B. Acta Neurol Scand (2000) 101:359-371).
While its mechanism of action is incompletely understood, current evidence suggests that gabapentin does not directly interact with GABA receptors in many neuronal systems, but rather modulates the activity of high threshold calcium channels. Gabapentin has been shown to bind to the calcium channel α2δ ancillary subunit, although it remains to be determined whether this interaction accounts for its therapeutic effects in neuropathic pain.
In humans, gabapentin exhibits clinically effective anti-hyperalgesic activity against a wide ranging of neuropathic pain conditions. Numerous open label case studies and three large double blind trials suggest gabapentin might be useful in the treatment of pain. Doses ranging from 300-2400 mg/day were studied in treating diabetic neuropathy (Backonja, M., et al., JAMA (1998) 280:1831-1836), postherpetic neuralgia (Rowbotham, M., et al., JAMA (1998) 280:1837-1842), trigeminal neuralgia, migraine and pain associated with cancer and multiple sclerosis (Di Trapani, G., et al., Clin Ter (2000) 151:145-148; Caraceni, A., et al., J Pain & Symp Manag (1999) 17:441-445; Houtchens, M. K., Multiple Sclerosis (1997) 3:250-253; see also Magnus, L., Epilepsia (1999) 40:S66-S72; Laird, M. A. and Gidal, B. E., Annal Pharmacotherap (2000) 34:802-807; Nicholson, B, et al., supra).
Ziconotide (Prialt®; SNX-111) is a synthetic analgesic derived from the cone snail peptide Conus magus MVIIA that has been shown to reversibly block N-type calcium channels. In a variety of animal models, the selective block of N-type channels via intrathecal administration of Ziconotide significantly depresses the formalin phase 2 response, thermal hyperalgesia, mechanical allodynia and post-surgical pain (Malmberg, A. B. and Yaksh, T. L., J Neurosci (1994) 14:4882-4890; Bowersox, S. S., J Pharmacol Exp Ther, (1996) 279:1243-1249; Sluka, K. A., J Pharmacol Exp Ther (1998) 287:232-237; Wang, Y-X., et al., Soc Neurosci Abstr (1998) 24:1626).
Ziconotide has been evaluated in a number of clinical trials via intrathecal administration for the treatment of a variety of conditions, including post-herpetic neuralgia, phantom limb syndrome, HIV-related neuropathic pain and intractable cancer pain (reviewed in Mathur, V. S., Seminars in Anesthesia, Perioperative medicine and Pain (2000) 19:67-75). In phase II and III clinical trials with patients unresponsive to intrathecal opiates, Ziconotide has significantly reduced pain scores and in a number of specific instances resulted in relief after many years of continuous pain. Ziconotide is also being examined for the management of severe post-operative pain, as well as for brain damage following stroke and severe head trauma (Heading, C., Curr Opin CPNS Investigational Drugs (1999)1:153-166). In two case studies Ziconotide has been further examined for usefulness in the management of intractable spasticity following spinal cord injury in patients unresponsive to baclofen and morphine (Ridgeway, B., et al., Pain (2000) 85:287-289). In one instance Ziconotide decreased the spasticity from the severe range to the mild to none range with few side effects. In another patient, Ziconotide also reduced spasticity to the mild range although at the required dosage significant side effects including memory loss, confusion and sedation prevented continuation of the therapy.
T-type calcium channels are involved in various medical conditions. In mice lacking the gene expressing the α1G subunit, resistance to absence seizures was, observed (Kim, et al., supra). Other studies have also implicated the α1H subunit in the development of epilepsy (Su, H., et al., J Neurosci (2002) 22:3645-3655). There is strong evidence that some existing anticonvulsant drugs, such as ethosuximide, function through the blockade of T-type channels (Gomora, J. C., et al., Mol Pharmacol (2001) 60:1121-1132).
Low voltage-activated calcium channels are highly expressed in tissues of the cardiovascular system. Mibefradil, a calcium channel blocker 10-30-fold selective for T-type over L-type channels, was approved for use in hypertension and angina. It was withdrawn from the market shortly after launch due to interactions with other drugs (Heady, et al., 2001). Growing evidence suggests T-type calcium channels may also be involved in pain. Both mibefradil and ethosuximide have shown anti-hyperalgesic activity in the spinal nerve ligation model of neuropathic pain in rats (Dogrul, et al., 2003).
U.S. Pat. No. 5,646,149 describes calcium channel antagonists of the formula A-Y—B wherein B contains a piperazine or piperidine ring directly linked to Y. An essential component of these molecules is represented by A, which must be an antioxidant; the piperazine or piperidine itself is said to be important. The exemplified compounds contain a benzhydril substituent, based on known calcium channel blockers. U.S. Pat. No. 5,703,071 discloses compounds indicated to be useful in treating ischemic diseases. A mandatory portion of the molecule is a tropolone residue, with substituents such as piperazine derivatives, including their benzhydril derivatives. U.S. Pat. No. 5,428,038 discloses compounds indicated to exhibit a neural protective and antiallergic effect. These compounds are coumarin derivatives which may include derivatives of piperazine and other six-membered heterocycles. A permitted substituent on the heterocycle is diphenylhydroxymethyl. Thus, approaches in the art for various indications which may involve calcium channel blocking activity have employed compounds which incidentally contain piperidine or piperazine moieties substituted with benzhydril but mandate additional substituents to maintain functionality.
Certain compounds containing both benzhydril moieties and piperidine or piperazine are known to be calcium channel antagonists and neuroleptic drugs. For example, Gould, R. J., et al., Proc Natl Acad Sci USA (1983) 80:5122-5125 describes antischizophrenic neuroleptic drugs such as lidoflazine, fluspirilene, pimozide, clopimozide, and penfluridol. It has also been shown that fluspirilene binds to sites on L-type calcium channels (King, V. K., et al., J Biol Chem (1989) 264:5633-5641), and also blocks N-type calcium current (Grantham, C. J., et al., Brit J Pharmacol (1944) 111:483-488). In addition, Lomerizine, as developed by Kanebo K K, is a known calcium channel blocker. However, Lomerizine is not specific for N-type channels. A review of publications concerning Lomerizine is found in Dooley, D., Current Opinion in CPNS Investigational Drugs (1999) 1:116-125.
U.S. Pat. Nos. 6,011,035; 6,294,533; 6,310,059; and 6,387,897 describe selective N-type calcium channel blockers that were designed based on the recognition that the combination of a piperazine or piperidine ring coupled through a linker to a benzhydril moiety results in effective calcium channel blocking activity.
GB 1,281,188 describes 1-substituted-3-amidopyrrolidines with analgesic activity. These compounds have single phenyl groups at R and R2.
U.S. Pat. No. 4,785,119 describes a process for synthesizing certain simple 3-aminopyrrolidine compounds which contain phenyl or diphenyl linked to pyrrolidine by alkylene linkers of 1 or 2 carbon in length. These compounds are described as building blocks for construction of useful products in chemical, pharmaceutical, and agricultural industries, in particular for making certain antibacterial products.
U.S. Pat. No. 6,468,998 describes pyrrolidine compounds with 5,HT2 antagonist activity. Some of the compounds contain benzhydril linked to the 3-amino group, but only by C═O.